Process for extraction of β-glucan from cereals and products obtained therefrom

ABSTRACT

A process for obtaining β-glucan from cereal grain, such as barley and oats. A β-glucan product obtained by the process. Uses of the β-glucan product as a food ingredient and for treating various diseases or disorders. The process includes the steps of forming flour from the cereal grain, mixing the flour with water to form a slurry of a process for obtaining β-glucan from cereal grain including forming flour from the cereal grain, mixing the flour with water to form a slurry of an aqueous solution of β-glucan and a solid residue, separating the aqueous solution from the solid residue, and removing water from the aqueous solution by evaporation or ultrafiltration or combinations thereof to form a β-glucan containing gel or solid.

FIELD OF INVENTION

This invention relates to a novel process for the extraction of β-glucanfrom cereals, such as barley and oats. The invention also relates toβ-glucan products obtained from the process. The invention furtherrelates to uses of those products as food ingredients and therapeuticagents.

BACKGROUND

The term “β-glucan” refers to those polysaccharides which compriseD-glucopyranosyl units which are linked together by (1→3) or (1→4)β-linkages. β-Glucans occur naturally in many cereal grains such as oatsand barley. The molecular weight of β-glucan molecules occurring incereals is typically 200 to 2000 kiloDaltons.

β-Glucan is desirable as a food additive, for example, to impart texture(“mouth feel”) to foods or useful as edible films for food coatings.β-Glucan may also be used to add bulk to foods and has the advantage ofhaving a neutral flavour.

β-Glucan is also desirable as a therapeutic agent. There is evidencethat β-glucan can lower serum cholesterol levels, heal wounds, moderateglycaemic response, and alleviate constipation. β-Glucan can activelybind to specific cell receptors and therefore may be useful for thetreatment of a wide variety of disorders or diseases.

The known methods for extracting β-glucan from cereal grains, such asoats and barley, involve several steps. Firstly, the cereal grain ismilled to a flour prior to extracting β-glucan from the flour using warmor hot water or an aqueous alkali solution. The milling step facilitatesrelease of the β-glucan from the cereal. The aqueous extract of β-glucanis then separated from the solid flour residue. Finally, the β-glucan isrecovered from the extract.

The known methods of recovering the β-glucan from the aqueous extractinclude precipitation of the β-glucan using a water miscible solvent,such as alcohol, or by freezing and then thawing the extract to give aprecipitate of β-glucan which can be recovered by filtration orcentrifugation. The extraction of the β-glucan itself from the cereal isnot generally a costly process. However, the recovery of the β-glucanfrom the extract is costly. This is due to the large amounts of waterthat must be removed to give solid β-glucan.

In addition, it is difficult to control the molecular weight of theβ-glucan product obtained from known processes. High molecular weightβ-glucan is preferable for certain uses. For example, high molecularweight β-glucan is preferable for moderating glycaemic response and forlowering serum cholesterol levels. On the other hand, low molecularweight β-glucan may be preferable as a food additive. For example, lowmolecular weight β-glucan can form a gel having beneficial texturalproperties for processed foods.

In order to obtain a high molecular weight β-glucan product, previousmethods of β-glucan extraction from cereals have required that enzymespresent in the cereal be deactivated prior to the extraction step. Theenzymes are responsible for lowering β-glucan molecular weight and aredeactivated either by treating the flour with boiling ethanol/watermixtures or by treating the flour with an aqueous acid solution.

SUMMARY OF INVENTION

It is an object of this invention to provide a process for extractingβ-glucan from cereals and to provide a β-glucan product obtained fromthe process, or at least to provide a useful alternative process orproduct.

In one aspect of the invention there is provided a process for obtainingβ-glucan from cereal grain including:

-   -   forming flour from the cereal grain;    -   mixing the flour with water at a temperature below approximately        65° C. to form a slurry of an aqueous solution of β-glucan and a        solid residue;    -   separating the aqueous solution from the solid residue;    -   removing water from the aqueous solution by evaporation or        ultrafiltration or combinations thereof to give a concentrated        aqueous solution of β-glucan; and    -   forming a β-glucan gel from the concentrated aqueous solution of        β-glucan.

Although the cereal used may be any cereal containing β-glucan, thepreferred cereal of the invention is barley or oats.

It is preferred that the gel is formed from the aqueous solution ofβ-glucan using any combination of the following steps: shearing,heating, cooling and freezing the solution. Shearing the solution may beby stirring the solution or by passing the solution down a pipe. Thesolution may also be heated and cooled to induce the formation of a gel.It is also preferred that the gel, once formed, is washed with water toremove starch or protein or starch or protein that may have beenhydrolysed. The gel may also be frozen, for example by extrusion into abath containing an aqueous solution of a salt where the temperature ofthe bath is below 0° C. The frozen gel is removed from the bath and thenthawed to give a more compact gel which is more readily isolated byfiltration. The gel may then be dried by, for example, spray drying orhot roller drying.

Preferably the step of milling the flour is carried out under dryconditions to enable the removal of starch from the cereal. Starchgranules can be removed from the milled flour by sieving or by airclassification. Alternatively, the cereal may be milled in the presenceof either cold water or a mixture of ethanol and water to facilitate theremoval of starch by standard methods.

The invention therefore also provides a starch rich fraction obtainedfrom the process of this invention and useful as an ingredient inprocessed foods, for malting, or as a feed for animals.

It is preferred in the extraction step that the flour is mixed withwater at a temperature greater than 45° C. but less than approximately60° C.

Preferably the aqueous solution of β-glucan is separated from the solidresidue by centrifugation or by filtration.

The invention therefore also provides a solid residue obtained from thisprocess and useful as an ingredient in processed foods, for malting, oras a feed for animals.

The β-glucan is recovered from the aqueous solution by firstlyconcentrating the aqueous solution of β-glucan. Concentration of theaqueous solution may be by evaporation, for example thin-filmevaporation, or ultrafiltration, to form a concentrated aqueoussolution. A β-glucan gel can be formed from this solution. The gel maybe washed with water to remove impurities and then dried, for example byspray drying or hot roller drying, to obtain a β-glucan solid.

Prior to concentrating the aqueous solution of β-glucan, it ispreferable to remove starch and/or protein impurities. Protein may beremoved by heating the aqueous solution to above about 70° C. causingthe protein to precipitate which can then be removed by filtration or bydecanting or by centrifugation.

Alternatively, protein may be removed by adding a protease to theaqueous solution followed by ultrafiltration of the degraded protein.Another method of removing protein is to add a flocculant such as acarrageenan, for example κ-carrageenan. Starch may also be removed byadding a starch degrading enzyme, such as an α-amylase, to the aqueoussolution followed by ultrafiltration to remove the degraded starch.

It is preferable during β-glucan extraction to add an enzyme to reducethe average molecular weight of the β-glucan. The enzyme is preferably acellulase (for example, E.C. 3.2.1.4).

It is also preferable during β-glucan extraction to degrade anyarabinoxylans present by adding an arabinoxylan degrading enzyme, forexample, a xylanase.

The invention also provides a β-glucan product produced by a process ofthis invention.

The invention further provides a composition containing β-glucanobtained by a process of this invention.

The invention also provides a method for lowering serum cholesterollevels in an animal including administering to the animal a β-glucanproduct obtained by a process of this invention.

The invention also provides a method for healing a wound in an animalincluding administering to the animal a β-glucan product obtained by theprocess of the invention.

The invention also provides a method for moderating glycaemic responsein an animal including administering to the animal a β-glucan productobtained by the process of this invention.

The invention also provides a method for alleviating constipation in ananimal including administering to the animal a β-glucan product obtainedby the process of this invention.

The invention also provides a method for stimulating the immune systemin an animal including administering to the animal a β-glucan productobtained by the process of this invention.

The invention also provides a food ingredient containing a β-glucanproduct obtained by the process of this invention.

The invention further provides an edible film as a food coating preparedusing β-glucan obtained by the process of this invention.

DETAILED DESCRIPTION

β-Glucan occurs naturally in a wide variety of cereals. The process ofthis invention is not limited to any particular cereal. However,preferred cereals are barley and oats.

The process of this invention can be varied to give different β-glucanproducts. The physical properties of a β-glucan product are dependentprincipally on the average molecular weight of the β-glucan moleculesand the conformation of the β-glucan molecules. High molecular weightβ-glucan is β-glucan having an average molecular weight greater than5×10⁵ Daltons. Low molecular weight β-glucan is β-glucan having anaverage molecular weight in the range of 5×10³ to 2×10⁵ Daltons.

β-Glucan products can form a gel in water. The ease with which aβ-glucan product forms a gel depends on the average molecular weight ofthe β-glucan and also depends on the manner in which a solution ofβ-glucan extracted from cereal grain is processed.

High molecular weight β-glucan is desirable for certain therapeutic usesbecause of its high viscosity in aqueous solution. The moderation ofglycaemic response and the lowering of serum cholesterol levels can beeffected using β-glucan of high molecular weight. However, enzymes knownto degrade β-glucan from high molecular weight β-glucan to low molecularweight β-glucan are known to be present in cereal grains. Therefore,known methods of obtaining a β-glucan from cereals have required anenzyme deactivation step, such as treatment with boiling ethanol/watermixtures or by treatment with an aqueous acid solution.

However, it is known that in some cereals, particularly barley, theβ-glucan degrading enzymes are present in the husk and outer layers ofthe grain. Thus, removal of the husk and outer layers of the grain bypearling leaves a cereal grain which has little or no β-glucan degradingenzyme present. In addition, the outer layers of the grain (the aleuroneand sub-aleurone layers) are depleted in β-glucan. The pearled grain istherefore enriched in β-glucan relative to unpearled grain.

During aqueous extraction of β-glucan from unpearled grain, colour,flavour, and enzymes from the husks of the grain can appear in theextract. Following further processing, this can result in a β-glucanproduct having an unacceptable colour or flavour, or being degraded bythe enzymes. Pearling of the grain removes the husks and outer layersand therefore minimises any undesirable colour or flavour of theβ-glucan product.

During mixing of the flour with water to extract the β-glucan, the watermay be at any temperature in the range of 25 to 65° C. However, thetemperature of the water is preferred to be approximately 45 to 60° C.Preferably the pH of the mixture is in the range 2 to 10.

Starch is the major constituent of the grain and occurs as smallgranules within the grain. β-Glucan occurs within the cell walls of thegrain which surround the starch granule. The complete or partial removalof starch from flour obtained from the grain would therefore result in afraction enriched in β-glucan. An enriched β-glucan fraction has thefollowing benefits. Firstly, there would be less solid material toremove after the extraction was complete. The extract would contain moreβ-glucan for a given volume of water used. Therefore, less concentrationof the extract would be required. Finally, less starch would besolubilised during the extraction since there is less starch in theflour from which the β-glucan is extracted.

Various methods are known for complete or partial removal of starch fromcereal grain. These include dry milling and wet milling. Wet millingwith water has a disadvantage since about 30–50% of the cell wallβ-glucan is soluble in water at a temperature of 25° C. However, only10–20% of the cell wall β-glucan is soluble in ice-cold water.Similarly, little of the cell wall β-glucan is soluble in ethanol orethanol/water mixtures or aqueous solutions of certain salts. Therefore,for wet milling, it is preferable to use cold water or ethanol/watermixtures or aqueous solutions of certain salts.

Dry milling may be used for removing starch. A large proportion of thestarch can be removed from dry flour by sieving or air classification.The cell wall material containing the β-glucan mostly occurs asparticles which are larger than the starch granules after milling.Consequently, the starch granules will pass through the sieve while cellwall material will be retained. Air classification will separate out thedense starch granules from the cell wall material. However, it is to beunderstood that these methods of separation are not 100% efficient andthat the starch fraction will contain some cell wall material and thecell wall material will contain some starch.

The β-glucan from the enriched β-glucan material can now be extractedusing hot water. Since there are little or no β-glucan degrading enzymesleft in the grain, it can be useful to add an enzyme, preferably acellulase, to the extraction solution to partially degrade the β-glucanin a controlled fashion. This also helps release the β-glucan from theenriched material. It can also be advantageous to add an arabinoxylandegrading enzyme, preferably a xylanase, since these degrade unwantedarabinoxylans in the extract, decreasing the extract viscosity,increasing the yield of extract after separation from the solids, andalso helping in the release of the β-glucan from the enriched flour.

After extraction the solids are preferably removed by centrifugation.The extract can be concentrated at this stage by evaporation of all orsome of the water. Such techniques for this are well known and includethin-film evaporation to obtain a concentrated β-glucan solution, andspray drying or hot roller drying to obtain a β-glucan containing solid.

The final product from this process contains protein and starch. In somecases this less pure form of β-glucan may be the preferred product.However, it may be desirable to remove the starch and/or protein priorto water evaporation to obtain a product of higher purity. Starch can bedegraded using a starch degrading enzyme, preferably α-amylase, andprotein can be degraded by a protein degrading enzyme, that is, aprotease. The degraded starch and protein can then be removed from theextract and the extract concentrated by ultrafiltration. It is alsopossible to precipitate the protein by heating the extract above about70° C. The precipitated protein can then be separated from the solutionof the extract. Heating the extract above 70° C. has the advantage ofalso destroying any remaining enzyme activity and sterilising theextract.

Heating the extract above about 70° C. appears to inhibit gel formation.Heated extracts appear not to form a precipitate when frozen and thawednor do they gel readily, that is, within a few hours. However, gelationcan be induced by the following methods, either alone or in combinationwith other methods. Resting the solution for a period of time, shearingthe solution for a period of time, cooling the solution for a period oftime, heating the solution for a period of time, and freezing thesolution for a period of time. Generally it is easier to induce gelationwith more concentrated solutions, especially those containing lowmolecular weight β-glucan.

Inducing gelation at this stage of the process has several advantagesover the technique of freezing and then thawing the solution. Theexpense of freezing in some cases can be avoided. Where freezing isstill required the solutions are more concentrated, thus decreasing thecost of freezing.

Following gelation it may be preferable to freeze the gel, for exampleby extrusion into a bath containing a salt, where the bath is at atemperature of less than 0° C. The gel is then recovered and thawed togive a compact gel which can be more easily filtered.

Finally after gelation has been induced it can be advantageous to washout the hydrolysed or unhydrolysed starch and protein contaminants fromthe β-glucan gel before the gel is dried to obtain a β-glucan enrichedgel.

The starch rich fraction obtained from sieving or air-classificationcould be a valuable product useful in baked and processed foods.Similarly, after the extraction of the β-glucan from the grain the wetsolids remaining contain significant amounts of β-glucan. These wetsolids could be dried and used in processed foods. The β-glucan in thedried solids could have useful texturising properties in a variety ofprocessed foods. It is also possible that the starch fraction or the wetsolids could be used for malting or sold as feed for animals.

The invention is described with reference to the following examples butis not to be construed as limited thereto.

In the examples all β-glucan contents were determined using the MegazymeMixed-Linkage Assay Procedure and the McCleary method or a modificationof the McCleary method. The starch and malto-oligosaccharide contentswere determined using the Megazyme Total Starch Assay Procedure or amodification of this procedure (Megazyme International Ireland Ltd, BrayBusiness Park, Bray, Co. Wicklow, Ireland)

EXAMPLE 1

Barley grain (50 g) was pearled from 40 to 60% and then finely milled ina Kenwood mixer with milling attachment. The milled grain was sievedthrough sieves of sizes 150, 90 and 63 μm. The coarse fraction left onthe sieve was further ground with a mortar pestle and sieved again.Yields, percentage and absolute β-glucan contents for each fraction areshown in Table 1.

TABLE 1 Fraction Sieve size/μm Yield/g β-glucan content % β-glucancontent/g Coarse >150 12.9 7.61 0.981 Medium 150 → 90  6.06 12.53 0.760Fine 90 → 63 2.32 11 .55 0.268 Very fine  <63 25.72 0.85 0.218

The very fine fraction was the largest fraction sieved but containedonly small amounts of β-glucan. The medium and fine fractions bothcontained about 12% β-glucan. Of the sieved flour fraction 83% of theβ-glucan occurred in the fraction that sieved between 150 and 60 μm.

EXAMPLE 2

The release of β-glucan from the medium sieved fraction obtained inExample 1 was determined in the presence of various enzymes: a cellulase(Trichoderma reesei species from Sigma, 6.3 U/ml), xylanase (Shearzyme™from Novo Nordisk, activity unknown) and protease (Alaclase™ from NovoNordisk, 2.4 AU/g). The medium sieved fraction (1 g, see Example 1) wasadded to various combinations of enzymes (see Table 2) in water (7 ml)and the mixture was heated for 1.5 h at 50° C. to extract β-glucan. Theβ-glucan extract was separated from the solids by centrifuging at 3500rpm for 10 min and then frozen. After thawing, the yield of precipitated(ppt) β-glucan was determined. The results are shown in Table 2.

TABLE 2 Yield of Yield of ppt T. reesei Shearzyme Alaclase extract /mlβ-glucan/g 10 ul 10 ul 10 ul 5.67 0.061 5 ul 10 ul 0 5.44 0.061 0 10 ul10 ul 5.73 0.051 5 ul 0 0 5 0.03

All the enzymes appeared to be effective in increasing the yield of pptβ-glucan but the Shearzyme™/cellulase combination appeared to be mosteffective. The yield of extract after centrifuging was improved byadding Shearzyme™.

EXAMPLE 3

A flour enriched in β-glucan was prepared by sieving a barley pollardflour. The β-glucan was extracted from the flour by heating a mixture ofthe flour (2 g) with water (10 ml) to which had been added cellulase (10μL, Trichoderma reesei species from Sigma, 6.3 U/ml) at 50° C. for 30min. The extract (6.1 ml) was separated from the solids by centrifugingat 3000 rpm for 15 min. The extract was then heated on a boiling waterbath for 5 min to, precipitate protein, which was removed bycentrifugation. The extract was evaporated to dryness by rotaryevaporation, which produced a glassy film containing about 53% β-glucan.

EXAMPLE 4

A barley pollard flour 10 g was mixed with water (50 ml) and heated at50° C. for 1 h. The extract was separated from the solids bycentrifuging at 3000 rpm for 10 min. This yielded 30 ml of extract. Theextract was then heated to 95° C. for 10 min and the protein thatprecipitated was removed on a centrifuge. The extract was concentratedon rotary evaporator to about 25% of its original volume. The extractwas then stirred rapidly for 2 min to induce shearing and then restedfor 5 min. This procedure was repeated 6 times before the extract wasfrozen for 12 h. No precipitate formed on thawing. Over a period of daysthe solution slowly thickened. After 2 days the solution was frozen andthawed again. This produced a precipitate, which was filtered, washedwith water and dried. The yield was 0.16 g.

EXAMPLE 5

A pollard flour (30 g) was mixed with water (150 ml) containingShearzyme™ (10 μL, Novo Nordisk, activity unknown,) and cellulase (50μL, Trichoderma reesei species from Sigma, 6.3 U/ml). The mixture washeated at 50° C. for 1.5 h. After 30 min the mixture was found to bereasonably free flowing. A β-glucan extract was recovered from themixture by removing the solids on a centrifuge. The yield of extract was118 ml.

The extract was treated in a number of ways.

-   -   a) 25 ml of the extract was filtered through glass fibre then        treated with amylase (200 μL, Bacillus species Sigma, 3480U/ml)        for 1 h 30 min at 30° C. to hydrolyse the starch in the extract.        The extract was then heated at 90° C. for 15 min and centrifuged        (3,000 rpm 10 min) to remove protein and destroy amylase        activity. The liquid recovered was 23 ml. The extract was        dialysed overnight to remove hydrolysed starch. The extract was        then evaporated to an oil in a rotary evaporator and oven dried        at 80° C. The β-glucan content of the oven dried material was        about 57%.    -   b) 25 ml of the extract was heated at 90° C. for 15 min then        centrifuged to remove protein. The liquid recovered was 23 ml.        The extract was rotary evaporated to about half its original        volume and then dried as a thin film in an oven at 80° C. The        β-glucan content of the film was about 30%. Approximately 0.2 g        of the film was dissolved in 2 ml of water at 90° C. to form a        transparent solution. The solution was cooled in ice and stirred        to induce shearing and then rested. This was repeated several        times. After leaving overnight a gel had formed. The gel was        frozen. The thawed gel was washed with water and filtered and        dried. The gel filtered very readily on a #3 sintered glass        filter. This yielded 0.066 g of dried gel. The β-glucan content        of the dried gel was 87%.    -   c) 25 ml of the extract was filtered through glass fibre and        then treated with amylase (200 μL, Bacillus species Sigma,        3480U/ml) for 30 min at 30° C. to hydrolyse the starch in the        extract. The extract was heated at 90° C. for 15 min and        centrifuged (3,000 rpm 10 min) to remove protein and destroy        amylase activity. The extract (0.4 ml) was placed in an        ultrafiltration centrifugal filter unit (Millipore        Ultrafree-MC). The filter unit was centrifuged (13,000 rpm for        40 minutes) and about 0.07 ml of liquid was recovered which was        oven dried to a thin transparent film.

EXAMPLE 6

Barley pollard flour (30 g) was mixed with water (150 ml) containingShearzyme™ (10 μL, Novo Nordisk, activity unknown) and cellulase (50 μL,Trichoderma reesei species from Sigma, 6.3 U/ml). The mixture was heatedon a water bath at 50° C. for 1.5 h. After 30 min the mixture was foundto be reasonably free flowing. The solids were removed from the mixtureby centrifuging and the extract that remained was heated at 90° C. for15 min. The protein that precipitated was removed on a centrifuge. Theyield of extract was 118 ml. The extract was concentrated to 17 ml byrotary evaporation. A viscous solution remained which was heated to 90°C. and cooled and then heated to 70° C. and cooled. This caused thesolution to set rapidly to a soft gel, which was dispersed in water toremove soluble impurities, and then filtered and dried. The yield ofdried gel was 0.71 g. The β-glucan content of the dried gel was 80%. Thewashings from the gel were rotary evaporated to an oil and then ovendried. This yielded 0.9 g of a glassy material. The β-glucan content ofthe glassy material was 4%. Therefore it appears that about 94% of theβ-glucan was in the dried gel and only 6% in the gel washings obtainedby filtering the gel.

EXAMPLE 7

The following examples illustrates a novel method for removing starchwhich does not result in much solubilisation of β-glucan.

Removal of starch from the cell-wall material was accomplished byhomogenising in a Kenwood mixer, barley flour (4 g) with water that wassaturated with a salt, in this case sodium sulphate. The solution wasfiltered through a 55 μm nylon mesh. The slurry filtered well,indicating little or no solubilisation of the β-glucan. Remaining on thefilter was the enriched cell-wall fraction (1.75 g) which contained10.4% β-glucan.

EXAMPLE 8

A gel is formed by concentrating a β-glucan extract. Barley flour (25 g)was mixed with water (175 ml) and a xylanase (6.2 μL, Shearzyme fromNovo Nordisk, activity unknown) and cellulase (125 μL, Penisillumfunicolsum 0.1 mg /ml) was added. The extraction solution was heated at50° C. for 1 h. The extract was separated from the solids bycentrifuging at 3500 rpm for 10 min. The extract was then heated at 90°C. for 10 min to precipitate protein, which was removed by filteringthrough a glass fibre filter. The extract was concentrated to 1/10 itsoriginal volume and left overnight in the fridge to gel. After heatingthe gel to 65° C. and then cooling the gel, the gel was firmer.

EXAMPLE 9

More β-glucan can be extracted from finely ground flour then coarselyground flour. For each of the medium and the coarse flour fractionsprepared in example 1, the flour (0.2 g) was mixed with water (2 ml) towhich a xylanase (0.1 μL, Shearzyme from Novo Nordisk, activity unknown)and cellulase (5 μL, Penicillium funicolusum from Sigma, 10 μg/ml) hadbeen added. The extraction was continued at 50° C. for 1 h. The extractwas separated from the solids by centrifuging at 3500 rpm for 10 min.The β-glucan content of the extract was then measured. For the mediumflour fraction about 70% of the β-glucan in the flour was extracted,whereas for the coarse material only about 50% of the β-glucan wasextracted.

EXAMPLE 10

Barley flour (5 g ) was mixed with water (35 ml) to which a xylanase(Shearzyme from Novo Nordisk, activity unknown) and a cellulase(Celluclast from Novo Nordisk, 1500 NCU/g) had been added according tothe quantities given in Table 3. The extraction solution was heated at50° C. for 2 h. The extract was separated from the solids bycentrifuging at 3500 rpm for 10 min. The extract was then heated at 90°C. for 10 min to precipitate protein, which was removed by centrifuging.After a freeze/thaw of the extract the precipitate of β-glucan solids inthe thawed liquid was filtered, and washed with water then ethanol. Thesolids were dried and the viscosity of a 1% solution measured. Mw, theweight average molecular weight was estimated from the viscosity usingthe method of Böhm, N. and Kulicke, W-M. Carbohydr. Res. 315 (1999)293–301, and are shown in Table 3.

TABLE 3 Shearzyme Celluclast Relative added/μL added/μL viscosity Mw 20.2  49 19000 2 0.02 123 75000 2 0 300 194000 

The molecular weight thus can be altered by changing the quantities ofβ-glucan degrading enzyme added to the reaction mixture.

EXAMPLE 11

It is advantageous to use cold-water to wash out the starch and causeminimum solubilisation of the β-glucan. Barley flour (0.2 g) containing8.5% β-glucan was mixed with water (2 ml) at 4.5° C. for 2 h. Theextract was separated from the solids by centrifuging at 3500 rpm for 10min. From the β-glucan content of the extract it was calculated thatonly about 5% of the β-glucan in the flour was solubilised.

EXAMPLE 12

For maximum protein precipitation the pH of the extract should be nearthe isoelectric point of the protein. Barley flour (10 g) was mixed withwater (70 ml) and the extract mixture was heated at 50° C. for 1 h. Theextract was separated from the solids by centrifuging at 3500 rpm for 10min. A portion (5 ml) of the extract was taken and the pH adjusted to7.0 with NaOH solution (0.1 M). On heating to 95° C. no proteinprecipitation was observed.

EXAMPLE 13

To decrease the amounts of starch and maltodextrins in the extracts itis advantageous to deactivate partially or completely the nativeamylases in the flour, which improves the purity and gel properties ofthe β-glucan. Acid treatment and heating was found to be effective indeactivating the amylases.

A solution of the amylase was prepared by mixing barley flour (20 g)with water (200 ml) and immediately centrifuging the mixture. Thesupernatant was then filtered with Glass fibre (Watman GF/A) to removefines. The supernatant was then treated by adjusting the pH and heating.Amylase activity of the supernatant was measured by mixing an equalamount of the treated supernatant with a potato starch solution (1.5%)and recording the decrease in viscosity. Results are shown in Table 4.

TABLE 4 Treatment Viscosity Viscosity Viscosity Viscosity Code ofsupernatant after 1 min after 5 min after 10 min after 20 min A None 7063 59 55 B Heated at 95° C. 87 90 89 88 for 15 min C PH adjusted to 3.890 90 89 86 then heated at 50 ° C. for 25 min, pH then adjusted to 5.4.For a) the untreated supernatant showed significant enzyme activity.With b) heating at 95° C. destroyed amylase activity. For c) adjustingto pH = 3.8 and then heating at 50° C. destroyed most of the enzymeactivity. There was only a small decrease in the viscosity of the potatostarch solution after the pH of the supernatant was adjusted back to 5.4(pH = 5.4 is near the optimum pH for amylase activity).

EXAMPLE 14

Protein precipitation by addition of a precipitating agent such ascarrageenan can be useful for removing additional amounts of protein.This improves the purity and gelling properties of the β-glucan. Foroptimum protein precipitation the pH of the solution should be below theisoelectric point of the protein.

A flour from a pearled barley (5 g) was mixed with water (35 ml) towhich had been added a xylanase (2 μL, Shearzyme from Novo Nordisk) anda cellulase (0.05 μL. Celluclast from Novo Nordisk, 1500 NCU/g). DiluteHCl (200 μL, 0.1 M) and carrageenan (150 μL, 1%, Viscarin BF 136C fromFMC) was added. A brown precipitate forms which was removed bycentrifuging.

EXAMPLE 15

Sieved barley fractions were prepared enriched in β-glucan. Barley (5.1%β-glucan content) was pearled to a weight loses of 30%. The grain wasmilled on the finest setting of a Kenwood mixer fitted with a grain millattachment. Of the flour formed, 5 g was hand sifted through twosuccessive sieves containing a 150 and 63 micron mesh. The coarsematerial retained as the over on the 150 micron sieve was ground in amortar and pestle until most past through the 150 micron sieve. Threefractions were obtained as shown in Table 5.

TABLE 5 Sieve β-glucan β-glucan % Code size/micron Yield content/% oftotal a >150 0.20 9.4 9 b 150 → 63 0.83 16.8 65 c  <63 3.33 1.7 26 Fromtable 5 it can be seen that 65% of the β-glucan was concentrated in thefraction over the 63 micron sieve and that the β-glucan content of thisfraction was about 16%.

EXAMPLE 16

The stability of a gel that had been frozen was tested by repeatedwashings with water. The gel (4.9 g) was filtered on a 55 μm mesh toremove excess water and the filtrate retained. The gel was then washedwith water (10 ml) and the second filtrate retained. The β-glucancontent of the filtrates and gel were measured. Results shown in thetable indicate little solubilisation of β-glucan in the gel duringwashing.

TABLE 6 Sample β-glucan content/% % of total β-glucan First filtrate0.15  2% Second filtrate 0.014 0.7% Gel 11.7 97%

EXAMPLE 17

Amylase deactivation lessens the amount of maltose and starchsolubilised during extraction. Water (10 ml) was adjusted to pH=2.4 withHCl (˜0.65 ml, 1.0 M) and added to flour (10 g) milled from a pearledbarley. For this mixture the pH was found to be 2.8. The mixture washeated at 50° C. for 20 min on a water bath to deactivate the amylase.The pH of the mixture was then adjusted to 5.5 with NaOH (2 ml, 1.0 M).A xylanase (4 μL, Shearzyme Novo Nordisk) and a cellulase (0.1 μLCelluclast from Novo Nordisk) was added to the extraction mixture andthe extraction continued for 1 h. The mixture was then centrifuged at(3000 rpm, 5 min) and the supernatant retained. The solution was thenacidified with HCl (1.6 ml, 0.1 M) and κ-carrageenan (1.2 ml, 1%) wasadded. The precipitate that was formed was removed by centrifuging togive a bright solution. The solution was lyophilised to a whitish solid.The above experiment was repeated, but no enzyme deactivation step wasincluded.

The starch/malto-oligosaccharide content of the solids with and withoutamylase deactivation was 9% and 26%, respectively.

EXAMPLE 18

An extract was formed from flour obtained from pearled barley accordingto Example 17 (with amylase deactivation). The solution obtained afterprotein precipitation was rotary evaporated to an oil and left at 4° C.for 2 days. During this time the oil set to a gel, which was washedseveral times with water. The gel was pressed between paper towels toremove excess water. Solids content of the gel was 17% of which 75.6%was β-glucan. Some 50% of the β-glucan in the flour was recovered in thegel.

EXAMPLE 19

Barley (1000 kg) was pearled to produce pearl barley (700 kg). The pearlbarley was milled through two roller mills and a hammer mill and thenscreened to produce two flour fractions. The first fraction (420 kg)contained approximately 80% of the β-glucan. The second fraction (280kg) contained approximately 20% of the β-glucan. The second fraction wasdiscarded. The first fraction was divided into seven batches (each 69kg).

Each batch was mixed into warm water (1200 L) to give a mixture at atemperature of 50° C. Cellulase (0.5 ml, Celluclast from Novo Nordisk,1500 NCU/g) and Xylanase (60 ml, Shearzyme from Novo Nordisk, activityunknown) enzymes were added to the mixture which was stirred and heldfor 60 minutes. The mixture was then passed through a solid bowldecanter and a centrifugal clarifier to remove all insoluble material.The insoluble material was discarded.

The resulting liquid extract (900 L) was adjusted to a pH of 4.5.Amyloglucosidase enzyme (150 ml, AMG 300 L from Novo Nordisk, 300 ACU/g)was then added to hydrolyse any soluble starch. After the extract wasshown to be starch negative, it was heated to 95° C. for 15 minutes andthen centrifuged to remove the insoluble protein.

The extract from all seven batches was combined and filtered through adiatomaceous earth filter. The filtered extract was then concentrated ina triple effect falling film evaporator, followed by a single effectscraped surface evaporator, to approximately 12% total solids. Theconcentrate was then cooled at less than 0° C. for 24 hours to develop asuitable gel structure. The gel was then washed in cold water to removethe remaining soluble sugars and other soluble material.

The gel was recovered from the mixture using a centrifugal clarifier andthen dried in a spray drier to approximately 5% moisture to giveβ-glucan powder (14 kg). The powder was a fine, free flowing pale creamβ-glucan powder. The β-glucan content was approximately 85% on a drysolids basis and had a molecular weight of approximately 50,000d.

EXAMPLE 20

A liquid extract was prepared according to Example 19 above but wassubjected to ultrafiltration following filtration through thediatomaceous earth filter, rather than concentration in a triple effectfalling film evaporator.

The extract (500 L) was collected after diatomaceous earth filtrationand was purified and concentrated using an ultrafiltration membrane. Theextract was circulated through a spiral type ultrafiltration membrane.The membrane had an area of approximately 6.4 sq. metres and a nominalmolecular cut-off of 10kd. Circulation was continued until the volume ofthe circulate was reduced to 100 L. Water (100 L) was added and thecirculation was continued until the volume was reduced again to 100 L.At the end of the process, 80% of the liquid had been removed aspermeate and the β-glucan purity had increased from 35% to 60% of totalsolids.

Although the invention has been described by way of example, it shouldbe appreciated that variations and modifications may be made theretowithout departing from the invention.

Furthermore, where known equivalents exist to specific features, suchequivalents are incorporated as if specifically set forth herein.

INDUSTRIAL APPLICABILITY

The β-glucan products of this invention are useful as food additives andas therapeutic agents. They provide desirable texture to foods, can beused as edible films for food coatings, and can be used as bulkingagents in foods. The products of the invention are also useful astherapeutic agents including agents for lowering serum cholesterollevels, healing wounds, moderating glycaemic response, alleviatingconstipation, and stimulating the immune system.

1. A process for obtaining a β-glucan gel from cereal grain including:forming flour from the cereal grain; mixing the flour with water at atemperature in the range of 25–65° C. to form a slurry of an aqueoussolution of β-glucan and a solid residue; separating the aqueoussolution from the solid residue; removing water from the aqueoussolution by evaporating or ultrafiltration or combinations thereof togive a concentrated aqueous solution of β-glucan; and forming a β-glucangel from the concentrated aqueous solution of β-glucan.
 2. A process asclaimed in claim 1 wherein the flour is formed by pearling the cerealgrain to remove the husk and outer layer of the cereal grain and thenmilling the pearled cereal grain.
 3. A process as claimed in claim 1wherein the pH of the water is adjusted either before or after mixingwith the flour.
 4. A process as claimed in claim 3 wherein the pH isadjusted to less than 4.0 and the slurry is then heated to greater than40° for at least 10 minutes to deactivate amylases in the flour.
 5. Aprocess as claimed in claim 4 wherein the pH is readjusted to greaterthan 4.0 after heating.
 6. A process as claimed in claim 1 wherein theflour is mixed with water at a temperature of 45° C. to 60° C.
 7. Aprocess as claimed in claim 1 wherein the flour is mixed with water at atemperature of 50° C. for 15 to 60 minutes.
 8. A process as claimed inclaim 1 further including adding an enzyme to the slurry to degrade anyarabinoxylans which may be present in the slurry.
 9. A process asclaimed in claim 1 where an enzyme is added to the slurry to assist therelease of β-glucan from the flour.
 10. A process as claimed in claim 1wherein the aqueous solution is separated from the solid residue bycentrifugation followed by decantation or by filtration.
 11. A processas claimed in claim 1 wherein an enzyme is added to the aqueous solutionto degrade starch.
 12. A process as claimed in claim 1 wherein an enzymeis added to the slurry or to the aqueous solution to reduce the averagemolecular weight of the β-glucan.
 13. A process as claimed in claim 1wherein a protease is added to the aqueous solution to degrade proteins.14. A process as claimed in claim 1 further including heating theaqueous solution to precipitate protein.
 15. A process as claimed inclaim 1 further including adding a flocculant to the aqueous solution toprecipitate protein.
 16. A process as claimed in claim 1 furtherincluding drying the gel.
 17. A process as claimed in claim 1 whereinthe β-glucan gel formation is conducted by shearing the concentratedaqueous solution, heating the concentrated aqueous solution, or coolingthe concentrated aqueous solution.
 18. A process as claimed in claim 1further including one or more of: freezing the gel and then thawing toincrease the density or compactness of the gel after the second formingstep; washing the gel with water to remove impurities, such as starch orprotein or fragments thereof after the second forming step; or removingimpurities from the aqueous solution by ultrafiltration and diafiltration after the separating step and before the second forming step.19. A process as claimed in claim 1 wherein the flour is formed bymilling the cereal grain under dry conditions and then removing starchgranules by sieving or by air classification.
 20. A process as claimedin claim 1 wherein the flour is formed by milling the cereal grain inthe presence of either cold water or a mixture of ethanol and water, oran aqueous salt solution, and then removing starch.
 21. A process asclaimed in claim 1 wherein the cereal is barley or oats.
 22. A processas claimed in claim 1, wherein the β-glucan gel formation is conductedby heating and then cooling the concentrated aqueous solution.